Summary A. PMS1 NTD:DNA To gain insight into the binding surface involved in the interaction of Pms1-NTD with DNA in the presence of a non-hydrolyzable ATP analog, a series of experiments involving limited proteolysis and OPF were performed. The results from these reactions were combined with mutation results from the Kunkel lab and have been mapped onto the solved structure of yPms1 NTD. We have now used these data to model the interaction of DNA with yPMS1 NTD. B. Anthrax Protective Antigen: Protective antigen (PA) undergoes a pH induced conformational change to form a pore through which edema factor and lethal factor are transported into the cell, resulting in cell death. This conformation change is characterized using oxidative protein footprinting. The prepore (pH 7.5) and pore (pH 5.5) structures are probed using OPF using laser photolysis flow cell system, the differentially modified residues are identified and mapped to the structure to identify solvent accessible residues of each conformation. Under physiological conditions PA is bound to a transmembrane anthrax toxin receptor (capillary morphogenesis gene 2, CMG2) associated with the cell membrane. In the previous experiments the lack of receptor and cell membrane may have serious implications in the structure of the PA complex. To overcome this limitation a lipoparticle system which incorporates a large number of the anthrax toxin receptors (CMG2) onto the surface of a stable nanoscale membrane particle is implemented. These non-infectious lipoparticles are derived from cells using retroviral structural proteins (GAG) and closely mimics in-vivo conditions for pH induced conformational change of PA. C. Technical advances in differential surface modification. A flow cell system was constructed for laser flash photolysis of protein solutions using a Nd:YAG laser. The laser photolysis system was developed to probe tertiary and quaternary protein structure by covalently labeling solvent accessible amino acids with hydroxyl radicals (oxidative protein footprinting, OPF). Hydroxyl radicals are produced by homolytic cleavage of hydrogen peroxide by ultraviolet laser photolysis. Laser photolysis has the advantage of fast oxidation of proteins in solution, faster than oxidation induced conformation changes thereby giving a snapshot of the proteins conformation for a given set of conditions. This is in contrast to previous work utilizing gamma irradiation to generate hydroxyl radicals from water in which the oxidation experiment required 30 to 40 minutes of irradiation exposure for sufficient oxidation, allowing time for conformation change and re-oxidation. The laser photolysis oxidation flow cell system has been characterized using a standard protein Beta-lactoglobulin (18 kDa) and is being applied to solving structural problems with the heptameric protein complex protective antigen from Bacillus anthracis. D. Structural characterization of Polymerase g interactions Polymerase g subunits, p140 and p55, were analyzed to characterize the interactions of the cysteine residues, which are known to be involved in formation of the polG complex. P140 and p55 were modified with NEM alone and as a complex. Surface mapping experiments were carried out by mass spectrometry to evaluate the accessibility of each cysteine in the apo and complex forms. Current experiments involve the modification of basic residues to characterize the DNA binding sites for p140 and p55.